The present invention relates to a stable esterase enzyme and to a process for the extraction thereof from a natural source, and to the use thereof for the cleaving of acyclic monoterpenyl esters into their monoterpenols. More particularly, the present invention relates to a process for the extraction of a novel and stable esterase from the inflorescence of palmarosa (Cymbopogon martinii), which can be used to cleave acyclic monoterpenyl esters into their monoterpenols.
Monoterpenyl esters are particularly widespread as the major oil components of several plant species and as minor components in many essential oils. Further more these influence the quality of essential oil distilled from such plants. As most of the aromatic plants are known by its high monoterpenyl alcohol contents in their oils, the presence of monoterpenyl esters in some aromatic plants alter the flavour characteristics of these oils, and thereby reducing their price value in the international market.
In conventional industrial processes, the monoterpenyl esters are hydrolyzed chemically by treatment with alkali (e.g. 10% alcoholic KOH) to produce the monoterpenols. The monoterpenols produced by such methods are not preferred by the perfumery industry, because alkali treatment alters their flavour characteristic. There is only one report in Mentha species, which describes the conversion of cyclic monoterpenyl acetates into their monoterpenols through cell suspension cultures [Werrmann and Knorr, J. Agric. Food Chem. 41: 517-520 (1993)], Bioconversion methods through plant/microbial enzymes have been frequently used for the production of the various secondary metabolites including essential oil constituents. Freely suspended and immobilized plant cells or enzymic preparations can be used for such bioconversion purposes. The employment of isolated plant enzymes was found to be the most promising because it produces a single compound through bioconversion. In addition, the precursors that cannot enter into living cells can be successfully converted using isolated enzymes. Plant enzymes are generally able to catalyze the reactions stereospecifically resulting in chirally pure products, and they can also perform regiospecific modifications that are not easily carried out by chemical synthesis or by microorganisms [Pras et al., Plant Cell, Tissue and Organ Culture 43: 117-121 (1995)]However, there are no such reports on the bioconversion of acyclic monoterpenyl esters into their monoterpenols.
Accordingly, the main object of the present invention is to provide a novel and stable esterase enzyme that hydrolyses monoterpenyl esters to yield acyclic monoterpenols.
It is another object of the invention to provide a process for the extraction of such novel and stable esterase enzyme from a natural plant source such as palmarosa inflorescence.
It is a further object of the invention to provide a stable esterase enzyme that is capable of cleaving monoterpenyl esters into their respective acyclic monoterpenols.
The novel esterase enzyme mentioned in this invention yields acyclic monoterpenols through hydrolysis of its corresponding monoterpenyl esters. The enzyme can also be useful to hydrolyze the oils containing mixture of acyclic monoterpenyl esters (such as citronella oil) into their monoterpenyl alcohols. The non-specific nature of this novel esterase can be exploited to convert acyclic monoterpenol esters into their corresponding monoterpenols commercially through immobilizing the enzyme.
It is another object of the present invention to develop a process for the extraction of a novel and stable esterase enzyme from a natural source, which is useful for cleaving acyclic monoterpenyl esters into their corresponding monoterpenyl alcohols.
It is a further object of the invention to provide a novel stable esterase that shows a linear increase in activity along with protein concentration.
It is yet another object of the invention to provide a site specific monoterpenyl ester hydrolase capable of removing the terminal ester group of acyclic monoterpenyl esters thereby producing monoterpenyl alcohols.
It is another object of the invention to provide a stable esterase derived from a natural source that is useful for the hydrolysis of acyclic monoterpenyl esters thereby recovering the delicate smell of monoterpenols.
Accordingly, the present invention provides a stable esterase extracted from a natural plant source.
In one embodiment of the invention, the plant tissues are selected from inflorescence of palmarosa, leaf tissues of lemongrass and some other aromatic plants.
In another embodiment of the invention, the esterase enzyme is a site specific monoterpenyl ester hydrolase capable of removing the terminal ester group of acyclic monoterpenyl esters.
In another embodiment of the invention, the esterase enzyme is stable when stored at 4xc2x0 C. for one week with only 40% loss of activity.
In yet another embodiment of the invention, the esterase enzyme from palmarosa inflorescence is most active in the alkaline pH range between 8.0-9.0 and temperature range between 20-40xc2x0 C.
In a further embodiment of the invention, optimum activity of the esterase enzyme is found at pH 8.5 and temperature 30xc2x0 C.
In yet another embodiment of the invention, the esterase enzyme has a linear catalytic rate of up to six hours of incubation at 30xc2x0 C.
The present invention also relates to a process for the extraction of a stable esterase from plant tissues from a natural source and useful for the cleaving of acyclic monoterpenyl esters into their corresponding alcohols, said process comprising: homogenizing the plant tissue in a cold extraction medium (1 g tissue/3 ml) consisting of 0.1 M NaPi buffer (pH 6.5) containing 50 mM sodium metabisulphite, 10 mM xcex2-mercaptoethanol, 10 mM ascrobic acid, 0.25 M sucrose and 1 mM EDTA-Na2, squeezing the slurry through four layers of muslin cloth, centrifuging at 15,000 xc3x97 g for 60-80 minute, adding purified amberlite XAD-4 resin to the supernatant (half of the tissue weight), keeping it for 4-6 minutes at 4xc2x0 C., filtering the slurry thus obtained through muslin cloth to get a clear supernatant, which is used as a esterase enzyme source.
In another embodiment of the process of the invention, the plant tissues from a natural source are selected from inflorescence of palmarosa, leaf tissues of lemongrass and some other aromatic plants.
In another embodiment of the invention, the buffer is selected from NaPi and Tris-HCl.
The invention also relates to a process for hydrolysis of the oil containing the mixture of acyclic monoterpenyl esters into their corresponding alcohols by using the novel stable esterase of the invention.
In another embodiment of the invention, the acyclic monoterpenyl esters that are hydrolysed are selected from geranyl acetate, geranyl formate and citronellyl acetate.
In a further embodiment of the invention, 75% of the geranyl acetate was hydrolyzed to its corresponding alcohol after 24 hours of incubation.